Characterization of genes involved in the iron acquisition system of multidrug-resistant Acinetobacter baumannii

Background: The high prevalence of virulence-associated genes observed in Acinetobacter baumannii isolates underscores the pathogenic potential of this bacterium. The presence of these genes confers enhanced survival, evasion of host defenses, and increased virulence. In this study, we investigate the presence and distribution of genes associated with virulence and assess the antimicrobial susceptibility patterns in clinical isolates of A. baumannii. Materials and method: This research focused on examining the 50 multi-drugs resistant (MDR) strains that were included in this investigation. The identification of these strains was validated using Oxa-51. The presence of the BauA and BasD genes was determined through conventional PCR techniques. Results: The results derived from Oxa-51 PCR confirmed the identification of all 50 selected strains of A. baumannii. Additionally, both the BauA and BasD genes were successfully identified in 82% of the MDR strains. Conclusion: Moreover, the varying antibiotic resistance patterns highlight the challenge in treating A. baumannii infections effectively. Strategies such as combination therapy, antimicrobial stewardship, and infection control measures should be considered to combat this multidrug-resistant pathogen.


Introduction
Acinetobacter baumannii is a Gram-negative bacterium that has become a major concern in healthcare settings due to its ability to cause infections that are difficult to treat [1], [2], [3].One of the key factors that contribute to its pathogenicity is its iron acquisition system, which allows the bacterium to obtain iron, an essential nutrient for bacterial growth and survival from its host [4], [5].Understanding the mechanisms and regulation of this system is crucial for developing effective strategies to fight A. baumannii infections [4], [5].The iron acquisition system in A. baumannii contributes to its pathogenicity by enabling the bacterium to proliferate and survive within the host [5], [6].The ability to acquire iron from the host provides A. baumannii with a competitive advantage over other bacteria, allowing it to establish infections and evade the host's immune responses [7]. A. baumannii employs several mechanisms to acquire iron from its environment, e.g., siderophores [7], iron-regulated outer membrane proteins (IROMPs), and Heme uptake systems [8].On the other hand, A. baumannii infections caused by antibiotic resistant strains are increasing and making treatment a challenge [9], [10].Understanding the molecular mechanisms of A. baumannii and its antibiotic resistance is crucial for developing effective treatment strategies [9].Two important genes, BauA and BasD, have been identified in A. baumannii and are believed to contribute to its virulence and antibiotic resistance [4], [5].The BauA gene encodes a protein responsible for the binding and uptake of ferric acinetobactin, a siderophore involved in iron acquisition [4].This allows the bacterium to absorb iron from the host environment, thereby promoting its survival.Additionally, the BauA protein has been implicated in biofilm formation, a crucial factor in the persistence and resistance of A. baumannii infections.By adhering to surfaces and forming biofilms, the bacterium can evade the immune system and resist antibiotic treatment [4], [11].The BasD gene codes for an enzyme involved in the production of acinetobactin, the aforementioned siderophore.Acinetobactin plays a vital role in the acquisition of iron.The activity of BasD is essential for the bacterium's ability to produce acinetobactin, thereby enhancing its virulence and resistance.Understanding the mechanisms underlying the regulation of BasD expression could potentially lead to the development of new therapeutic targets for combating A. baumannii infections [5].Indeed, the prevalence of antibiotic resistance in BauA and BasD positive strains were significantly greater compared to the equivalent susceptible isolates [5], [12]; even the antibiotic cross-resistant profile is found more often in MDR A. baumannii isolates which possess some virulence genes, such as BauA [5].This indicates that drug-resistant A. baumannii isolates seem to possess enhanced toxicity [5], making the identification of A.baumannii with virulence genes such as BauA and BasD is necessary.Hence, this study aimed to determine the prevalence and frequency of BauA and BasD genes in multi-drug resistant strains of A. baumannii.

Materials and methods
This detailed analysis, conducted as part of a research study, included a total of 50 multidrug-resistant (MDR) A. baumannii strains.These specific varieties were gathered from various divisions within ten educational medical facilities situated in Iran.The considered isolates studied here are associated with various units within the hospital, such as the intensive care unit (ICU), surgical department, neonatal intensive care unit (NICU), and others.Furthermore, there were instances where bacterially caused infections originated from different sources, e.g., blood, urine, and wounds.Initially, the confirmation of A. baumannii was achieved by amplifying the Oxa-51 gene using specific forward and reverse primers, 5'-TAATGCTTTGATCGGCCTTG-3' and 5'-TGGATTGCACTTCATCTTGG-3', respectively [3].The identification of the target genes was then performed through conventional PCR, under previously established experimental conditions.For DNA extraction, a boiling method was employed, and the extracted DNA samples were stored at -80ºC until the PCR analysis was conducted.The following primers were used for simultaneous gene duplication of BauA and BasD genes according to the results of the primer BLAST.NCBI (National Center for Biotechnology Information) [12].These primers possess the ability to detect and determine the presence of both the BauA and BasD genes, simultaneously.The primers sequencing are; Forward: 5'-CTCTTGCATG-GCAACACCAC-3' and Reverse: 5'-CCAACGAGACCGCTTAT-GGT-3' [5], [13].

Results
Our results indicate that the majority of these isolates were collected from the ICU.In terms of prevalence in this study, invasive catheters were commonly linked to these bacterial infections, followed closely by blood culture.The identification of all A. baumannii was confirmed according to the results of the PCR for the Oxa-51 gene.Moreover, the results of PCR and gel electrophoresis showed that in 82% of the MDR strains, both BauA and BasD genes were successfully detected.

Discussion
Iron acquisition is a crucial aspect of A. baumannii pathogenesis, enabling the bacterium to survive and cause infections in the host [4], [5].Understanding the mechanisms and regulation of iron acquisition in A. baumannii is essential for the development of effective therapeutic strategies.Further research in this field will provide valuable insights for combating A. baumannii infections and reducing their impact on healthcare settings [7], [13].The BauA and BasD genes in A. baumannii play crucial roles in promoting the bacterium's virulence and antibiotic resistance [5], [13].The BauA protein facilitates iron acquisition and biofilm formation, while the BasD enzyme is responsible for the production of acinetobactin.Both genes contribute to the bacterium's ability to survive and cause persistent infections [5], [10].Further research is needed to fully elucidate the mechanisms by which BauA and BasD influence antibiotic resistance, thus providing valuable insights for the development of effective treatment strategies against A. baumannii infections.
In the present study, BauA and BasD genes in MDR A. baumannii were present at a rate of 82%.Conversely, a study conducted in Iran by Porbaran et al [4] revealed a lower frequency of 15.2% and 12.5% for the BauA and BasD genes, respectively in A. baumannii.This disparity in frequencies could be attributed to differences in strain selection between the two studies.In the current study, MDR A. baumannii strains were chosen, while Porbaran et al. [4] selected A. baumannii strains with different patterns of antibiotic resistance.The BauA gene serves as one of the mechanisms for antibiotic resistance in A. baumannii, and it is evident that its frequency is higher in MDR strains.In China in 2018, the BauA and BasD genes were found to have a frequency of 78.3% and 95.7%, respectively in the MDR A. baumannii strain [5].The outcomes of that particular investigation [5] are closely comparable to those of the present study, since both studies focused on MDR strains.Additionally, it reinforces the notion that the presence of the BauA gene leads to antibiotic resistance [4].Porbaran et al. [4] also revealed a noteworthy correlation between the distribution of iron/siderophore-uptake genes (e.g., BauA) and antibiotic resistance.Furthermore, another study demonstrated a high frequency of the gene encoding BauA, particularly within multidrug-resistant isolates [13].This finding aligns perfectly with our data.The frequency of BauA and BasD genes was high in MDR A. baumannii which was included in this study.

Conclusion
This study provides valuable insights into the frequency of BauA and BasD genes in A. baumannii clinical isolates.The high prevalence of these genes emphasizes the need for enhanced surveillance and infection control measures to limit the spread of multidrug-resistant A. baumannii strains.Regional differences in gene frequencies indicate the importance of tailored intervention strategies based on the specific resistance mechanisms prevalent in different healthcare settings.Further research is warranted to explore the clinical implications of these findings and to develop effective strategies to mitigate the impact of A. baumannii antibiotic resistance.